Review Article

Human circadian rhythms: physiological and therapeutic relevance

of light and melatonin
Debra J Skene and Josephine Arendt

Addresses
Centre for Chronobiology, School of
Biomedical and Molecular Sciences,
University of Surrey,
Guildford GU2 7XH, UK
Correspondence
Professor Debra J Skene
E-mail: d.skene@surrey.ac.uk
This article was prepared at the invitation of
the Clinical Sciences Reviews Committee of
the Association for Clinical Biochemistry.
Abstract
Ocular light plays a key role in human physiology by transmitting time of day
information. The production of the pineal gland hormone melatonin is under the
control of the light–dark cycle. Its profile of secretion defines biological night and
it has been called the ‘darkness hormone’. Light mediates a number of non-
visual responses, such as phase shifting the internal circadian clock, increasing
alertness, heart rate and pupil constriction. Both exogenous melatonin and light,
if appropriately timed, can phase shift the human circadian system. These
‘chronobiotic’ effects of light and melatonin have been used successfully to alleviate
and correct circadian rhythm disorders, such as those experienced following travel
across time zones, in night shift work and in circadian sleep disorders. The
effectiveness of melatonin and light are currently being optimized in terms of time of
administration, light intensity, duration and wavelength, and melatonin dose and
formulation. The aim of this review is not to replicate information that has been
reported in a number of reviews of the human circadian timing system and the role
of melatonin and light, but rather to extract findings relevant to the field of clinical
biochemistry.
Ann Clin Biochem 2006; 43: 344–353

Introduction moving from a day shift to a night shift); can be a result

Most physiological (temperature, hormones, electro-
lytes) and behavioural (mood, alertness, sleep, perfor-
mance) measures exhibit daily rhythms. Circadian
rhythms are de¢ned as those rhythms that are endo-
genously generated, have a periodicity of approxi-
mately 24 h and whose rhythmicity persists when
environmental conditions (light, temperature, posture,
etc.) are kept constant. An endogenous circadian
timing system allows an organism to anticipate and
predict daily changes in the environment. Circadian
rhythms are generated by a pacemaker localized in the
hypothalamic suprachiasmatic nuclei (SCN); lesions of
these nuclei produce arrhythmicity. In normal condi-
tions, circadian rhythms are synchronized (entrained)
to the 24 h day primarily by the light--dark cycle.
Entrainment to the environmental light--dark cycle
allows the timing of daily activities (e.g. sleeping and
feeding) to be optimized. Circadian rhythm disorders
can be caused by a mismatch between external and
internal time (e.g. following £ight across time zones,
of circadian ‘dysentrainment’ (e.g. circadian rhythm
sleep disorders, advanced and delayed sleep phase
insomnia [ASPS and DSPS]); or can relate to dysfunc-
tional photic input to the SCN clock (e.g. in some types
of blindness).
The human circadian system
Human circadian rhythms
Examples of human circadian rhythms are core body
temperature, and secretion of cortisol, melatonin and
thyroid-stimulating hormone (TSH). In normal condi-
tions, these rhythms have a characteristic time of peak
and nadir and there is a set time interval (phase rela-
tionship) between the di¡erent rhythms (Figure 1). In
the absence of a light--dark cycle (such as experienced
by totally blind individuals or in sighted individuals
kept in very dim light conditions), circadian rhythms
become desynchronized from the 24 h day and ‘free-
run’at their intrinsic period length (t). Although there
is some debate as to the average period length (t) in

344 r 2006 The Association for Clinical Biochemistry

 Human circadian rhythms 345

humans, most studies agree that t is highly individual.
Studies in sighted people in dim light conditions and
forced desychrony experiments have reported average
t’s of approximately 24.2 h,1--3 whereas studies in
totally blind people with free-running circadian
rhythms have reported slightly longer t’s of 24.5 h.4--7
The reasons for this discrepancy in t are not yet known
but may relate to methodological di¡erences or the
(a) 60
concentration (pg/mL)
Plasma melatonin
di¡erences in light perception and previous light his-
tory between the study groups.
Measurement of circadian rhythms
In order to establish whether a daily rhythm in a phy-
siological parameter is endogenously generated (circa-
dian) or a result of changes in the environment
(exogenous), or both, ‘constant routine’ protocols are
performed (this is currently considered the gold stan-
dard).8 In constant routine studies, all possible known

confounding factors are kept ‘constant’ so that partici-

40
pants are required to remain awake; maintain a con-
stant posture (e.g. semi-recumbent); in constant
20 lighting conditions, usually dim light o10 lux (10 lux
is approximately the light given by a candle 1ft away
0 from an object; it is possible to read and see quite well
in dim light once the eyes have adapted); and receive
(b) 37.1 isocaloric meals hourly for at least 25 h. The contribu-
temperature (οC)

tion of the circadian system (endogenous) and the

environment (exogenous) to an observed rhythm can
Core body

36.9
also be quanti¢ed in ‘forced desynchrony’ experi-
36.7
ments.3 In these experiments, participants live on a 20
or 28 h day for at least 12 days. Forced desynchrony
protocols are designed to be able to separate the in£u-
36.5
ence of the sleep--wake cycle from the in£uence of the
(c) 60 circadian pacemaker.
Although these experiments are extremely demand-
Subjective alertness

ing on both participants and researchers, they reliably

100 = very alert)
(0 = not alert,

40
estimate the circadian component of an observed
rhythm. There are also confounding factors even to
20 forced desynchrony experiments (e.g. scheduling of
sleep length), and because of ¢nancial and resource
0 constraints (e.g. temporal isolation facilities are
required), these experiments are not always possible.
(d) 1.8 Fortunately, measurement of the endogenous melato-
Task performance

nin rhythm provides a relatively reliable surrogate

reaction time (ss)

1.6 way of assessing the timing of the internal circadian

clock in less-controlled conditions provided that cer-
1.4 tain precautions are taken (see later). Measurement of

1.2
Figure 1 Diagrammatic representation of the circadian
(e) 2.0
rhythms of plasma melatonin, core body temperature,
subjective alertness, task performance (reaction time, in
concentration (mmol/L)

1.8 seconds) and triacylglycerol from human beings held in

Triacylglycerol

constant routine conditions (i.e. awake, controlled light,

1.6 posture, activity and meals). The peak in the melatonin rhythm
(panel a), shown by the dotted line, and the low point of the
temperature rhythm (panel b) are within 1 h of each other. The
1.4
low point of the alertness and performance rhythms (panels c
and d, respectively) is shortly after the melatonin peak, and the
1.2 peak in triacylglycerol (panel e) is close to the melatonin peak.
1000 1400 1800 2200 0200 0600 1000 1400 Reproduced with permission from: Rajaratnam SM, Arendt J.
Clock time (h)
Health in a 24-h society. Lancet 2001; 358: 999–100572

Ann Clin Biochem 2006; 43: 344–353

346 Skene and Arendt
melatonin in plasma or saliva -- or measurement of its
major metabolite 6-sulphatoxymelatonin, usually in
urine -- can enable long-term monitoring of the human
circadian system in laboratory and real-life studies (see
later section).
Molecular clockworks
In recent years, major progress has been made in un-
derstanding the molecular mechanism underlying cir-
cadian oscillations within individual SCN cells. Clock
genes and clock-controlled genes are rhythmically ex-
pressed primarily via an intracellular transcriptional/
translational negative feedback loop.9,10 In brief, during
the day, transcription of a number of genes (Per1, Per2,
Per3, Cry1, Cry2 and Reverba) is activated by hetero-
meric complexes of CLOCK and BMAL1. This transcrip-
tion continues into the night until nuclear levels of PER
and CRY proteins become su⁄ciently high to repress
CLOCK/BMAL1 activation. Declining levels of PER/
CRY in the early morning then allows transcription of
the genes again and the cycle continues. This constitu-
tes the negative feedback limb of the oscillator core.
There is an auxiliary loop driven by Reverba (and possi-
bly Reverbb). The protein products, REVERBa and b,
suppress Bmal1 transcription, disappearance of RE-
VERB proteins allowing Bmal1 to peak at the end of
the night (positive limb). Rhythms in clock gene
expression have also been characterized in numerous
peripheral tissues (e.g. liver, heart, lung), providing
substantial evidence for the presence of peripheral
clocks.11,12 The question of how these peripheral clocks
interact with the central SCN clock (feedforward/feed-
back) and how they are synchronized (directly via
hormonal and/or neural pathways and/or indirectly
via behaviour [e.g. feeding]13) is presently a major
research question.
Circadian photoreception
Considerable advances have also been made regarding
the neuroanatomical pathways involved in the trans-
mission of light information from the retina to the
SCN (circadian photoreception). Both animal14,15 and
human16,17 studies have shown that, in addition to the
classical rods and cones, a novel photoreceptor system
is involved in mediating so-called ‘non-image forming’
visual functions. In addition to image formation, light
entering the eyes is responsible for a number of non-
image-forming responses, such as circadian resetting
and entrainment, suppression of nocturnal melatonin
production, elevation of core body temperature and
heart rate, increased alertness and performance, and
pupil constriction. The discovery of melanopsin,18 a no-
vel photopigment, and its localization in a network of
intrinsically photosensitive retinal ganglion cells,19
has provided a basis for understanding how the light
Ann Clin Biochem 2006; 43: 344–353
signal is captured and transmitted to the SCN clock, in
addition to other brain structures. This melanopsin
network, with some contribution from the rods and
cones, is thought to mediate the non-visual e¡ects of
light.20 Ocular light is the major time cue (zeitgeber)
responsible for synchronizing the human circadian
timing system. Although early work suggested that
‘non-photic’ time cues such as meals, exercise and the
sleep--wake cycle were important synchronizers of the
human circadian clock,21 today light is considered the
primary time cue.22 Evidence to support this view has
come from detailed studies of totally blind people, who
exhibit free-running circadian rhythms in spite of liv-
ing with strong social cues.5--7,23 The contribution of
non-photic time cues to circadian entrainment, how-
ever, cannot be ruled out entirely. The demonstration
of relative co-ordination in blind subjects with
free-running circadian rhythms23,24 and non-photic
entrainment in a few blind subjects4 suggests that
non-photic zeitgebers may have a weak e¡ect on the
circadian system. At present, however, the non-photic
stimulus or stimuli responsible for these e¡ects have
not been identi¢ed. The ability of appropriately timed
exercise25 or exogenously administered melatonin26
to shift circadian rhythms represents the best evidence
to date that indeed non-photic stimuli can a¡ect clock
timing. Future studies investigating how photic and
non-photic stimuli interact to determine circadian
phase are required.
Melatonin
Synthesis and metabolism
The pineal hormone melatonin is intimately linked
with the circadian timing system. The rhythm in mela-
tonin synthesis is controlled by the SCN via a multi-
synaptic pathway (SCN -- paraventricular nuclei [PVN]
-- superior cervical ganglia [SCG]) that terminates in
the release of noradrenaline from postganglionic SCG
¢bres in the pineal gland.27 The SCN regulates the
melatonin rhythm via GABAergic inhibition and
glutaminergic stimulation of the PVN.28,29 The rate-
limiting enzyme in the synthesis of melatonin is aryl-
alkylamine N-acetyltransferase (AANAT). It is likely
that the human AANAT enzyme is regulated primarily
at a post-transcriptional level, whereas in rodents the
key event appears to be cyclic adenosine monopho-
sphate (cAMP)-dependent phosphorylation of a tran-
scription factor that binds to the AANAT promoter.
Rapid decline in activity with light treatment at night
appears to depend on proteasomal proteolysis of AA-
NAT following dephosphorylation and removal of a
protective 14-3-3 protein.30,31 (Evidence from both
structural studies and sequence analyses support the
notion that the primary function of 14-3-3 proteins lies

in their preferential binding to phosphorylated sub-
strates. In humans, there are seven 14-3-3 isoforms (b,
g, e, z, Z, i, s).)30,31 Melatonin production peaks during the
dark phase in all species studied to date. Melatonin is
metabolized primarily in the liver via a cytochrome
P450-mediated hydroxylation into 6-hydroxymelato-
nin. It is then conjugated, primarily with sulphate to
form 6-sulphatoxymelatonin and, to a lesser extent,
with glucuronic acid.32,33
Role of endogenous melatonin
The role of endogenous melatonin in human physiol-
ogy is not clearly de¢ned. Although the e¡ects of exo-
genous melatonin have been extensively studied
(mainly at pharmacological doses),26,33,34 more exten-
sive studies with pinealectomized patients and devel-
opment of a speci¢c melatonin receptor antagonist for
human studies may help to clarify its role further.
Endogenous melatonin is a hormonal signal that gives
time of day (and time of year) information to the body.
Secreted at night in all species studied to date, it
appears to reinforce darkness-related behaviours such
as sleep initiation. An association between the rise in
melatonin production at night and the increase in sleep
propensity has been noted.35 In addition, abnormal
timing of melatonin in totally blind individuals has
been associated with increased napping.36 However,
whether these e¡ects of endogenous melatonin are
causal or merely correlational is not known.
Melatonin receptors
Melatonin receptors are high a⁄nity (picomolar KD)
G-protein (mainly Gi) receptors. Three melatonin
receptors have been cloned, namely mel1a (MT1), mel1b
(MT2) and mel1c, by Reppert and colleagues.37--39
Although melatonin receptors have been found in the
human SCN and pituitary stalk,40,41 relatively little
work has been done in the area, most likely because of
the lack of availability of human tissue. Further identi-
¢cation and localization of melatonin receptors may
provide some clues as to melatonin’s physiological
function in humans. In animals, melatonin receptors
in the SCN have been linked to suppression (MT1) and
phase shifting (not MT1, possibly MT2) SCN activ-
ity.42,43 In sheep, MT1 receptors in the pars tuberalis
mediate the photoperiodic regulation of prolactin pro-
duction.44 Melatonin receptors in the mediobasal
hypothalamus have been proposed to mediate
melatonin’s e¡ects on the gonadal axis of seasonal bree-
ders.45 Whether endogenous melatonin has a similar
role in prolactin production and gonadal development
in humans has long been speculated but de¢nitive
proof remains lacking. Exogenous melatonin has been
reported to a¡ect prolactin production in some clinical
Human circadian rhythms 347
studies.33,46--48 The e¡ect of melatonin administration
on gonadal hormones is more controversial. Most
recent studies with near-physiological doses of melato-
nin show no short-term e¡ects on gonadotrophins and
gonadal hormones.49,50 However, altered melatonin se-
cretion has been reported in a number of reproductive
pathologies (reviewed byArendt51,52).
Endogenous melatonin rhythm
The circadian rhythm of melatonin has characteristics
that can be used to measure the phase of the circadian
system. In normal 24 h entrained conditions, melato-
nin peaks at 02.00--03.00 h, rising approximately 4 h
earlier (22.00--23.00 h) and declining back to baseline
levels in the morning (09.00--10.00 h), while sleep
occurs 1--2 h after melatonin onset, ends 1--2 h before
melatonin o¡set and usually lasts 7 h. The timing of
the rhythm can be measured by estimating the melato-
nin onset, peak or o¡set (Figure 2). Although the time
of melatonin onset in dim light conditions has been
used frequently as a marker of circadian phase, it may
be better to measure both melatonin onset and o¡set as
there is evidence to suggest that these may be shifted
di¡erentially.53 Ideally, however, the whole melatonin
pro¢le should be measured. The amplitude of the
rhythm and the amount of melatonin produced (e.g.
area under the curve) is highly variable between indi-
viduals (thought to be related to the size of the pineal
gland54). However, within an individual, melatonin
production is fairly consistent.
Importance of measuring melatonin
The timing of the melatonin rhythm is today consid-
ered the most reliable marker of the timing of the circa-
dian clock. Control of the lighting environment and, to
a lesser extent, posture, is required for accurate melato-
nin measurement but, apart from this, of all possible
‘rhythm markers’, melatonin is least a¡ected by activity,
sleep, timing of meals, stress, menstrual cycle, etc. In
contrast, other circadian rhythms such as core body
temperature and secretion of cortisol have been shown
to be signi¢cantly a¡ected by these factors. Measure-
ment of melatonin and 6-sulphatoxymelatonin has
been successfully used to monitor circadian de-
synchrony in blind people in their home environ-
ments5,55 as well as in DSPS,26 in people working night
shifts56,57 and in people after crossing time zones.26
The timing of the melatonin rhythm in these studies
has provided important information as to the indivi-
dual’s circadian phase and has been used to optimize
the timing of light and melatonin in the treatment of
circadian rhythm disorders (discussed later on).
In addition to being a marker of circadian phase,
measurement of melatonin is also performed to provide
an assessment of pineal gland function. Conditions
Ann Clin Biochem 2006; 43: 344–353
348 Skene and Arendt

80
Duration
70

60

Plasma melatonin (pg/mL)

Acrophase (calculated peak time)
50 *

40

30 Mid-range crossing
* *
20
25% rise/fall
10 * *
Onset/offset
* *
0
1500 1700 1900 2100 2300 100 300 500 700 900 1100 1300 1500 1700

Clock time (h)

‘Biological night’

Figure 2 Normal profile of melatonin in plasma defining ‘biological night’ based on our laboratory normal values (n ¼ 133, healthy
men and women, all ages). Parameters of this profile and that of salivary melatonin and urinary 6-sulphatoxymelatonin that are used
to characterize the timing of the circadian clock are indicated, namely acrophase, duration, mid-range crossing, 25% rise and fall,
onset and offset of secretion. 6-Sulphatoxymelatonin acrophase is approximately 2 h after the plasma melatonin acrophase.
Redrawn from: Arendt J, Skene DJ. Melatonin as a chronobiotic. Sleep Med Rev 2005; 9: 25–39.26

in which pineal gland function might be compromised
include uveitis, various pineal tumours, disruption of
neural pathways to the pineal and/or the SCN (e.g. cer-
vical cord lesion, diabetic autonomic neuropathy).33,52
Increased melatonin concentrations have been
reported in conditions in which metabolism of melatonin
is compromised (e.g. liver cirrhosis) and in some repro-
ductive pathologies (e.g. amenorrhoea, hypogonado-
trophic hypogonadism).33,52 Numerous studies have
reported reduced melatonin production in other condi-
tions, e.g. breast cancer, sleep disorders (inconsistent),
depression, heart disease (reviewed byArendt51,52) and
Alzheimer’s disease.58 Because of the well-reported in-
ter-individual variation in melatonin concentrations as
well as an age-related decline,33,58 a cautious approach
to interpreting cross sectional studies should be
adopted. Longitudinal, within-subject studies are pre-
ferable to determine whether a condition a¡ects pineal
function or not.
Analytical measurement
The gold standard for measuring melatonin is gas chro-
matography--mass spectrometry (GCMS).59 For routine
measurement, radioimmunoassays (RIAs) have been
developed and validated using GCMS and these serve
as the most popular and widely used way of measuring
melatonin and its metabolite, 6-sulphatoxymelatonin.
The current assays and protocols have been recently
reviewed.60 Assay reagents, antibodies and assay kits
are available from a number of manufacturers, includ-
ing Stockgrand Ltd (www.stockgrand.co.uk), ALPCO
(www.alpco.com) and Buhlmann Laboratories
(www.buhlmannlabs.ch). Development of enzyme-
linked immunosorbent assays (ELISAs) in this area
has been slow but ELISAs for saliva melatonin and
6-sulphatoxymelatonin are currently available.
Factors affecting melatonin (reviewed by Arendt34)
Factors known to a¡ect the timing and production of
melatonin are several. The light--dark cycle has the
most signi¢cant a¡ect on melatonin timing and pro-
duction. Light is able to advance or delay the timing of
the melatonin rhythm, depending on when it is admi-
nistered. Light at night also has an acute suppressive
e¡ect on melatonin synthesis.61,62 Light transmitted
via the SCN--PVN--SCG pathway rapidly inhibits the
activity of AANAT, melatonin’s rate-limiting biosynthetic
enzyme, via proteasomal proteolysis (see previous sec-
tion). Thus, it is essential that the light/dark environ-
ment is controlled or, if control is impossible, at least
monitored, when measuring a person’s melatonin con-
centrations. Ideally samples should be collected from
subjects in constant dim light conditions (o10 lux at
eye level). Posture has also been shown to a¡ect the
measurement of plasma melatonin,63 thus ideally this
should also be controlled during the sampling period
(routinely subjects are asked to remain seated or semi-
recumbent). Drugs reported to interfere with melatonin

Ann Clin Biochem 2006; 43: 344–353

 Human circadian rhythms 349

measurements usually a¡ect the synthesis and/or Circadian rhythm disorders

metabolism of melatonin. Melatonin is synthesized
from serotonin via a b1-adrenergic mechanism, and to
a lesser extent an a-adrenergic mechanism. Drugs
known to interfere with these processes (e.g. serotonin
and noradrenaline reuptake inhibitors; a- and b1- ago-
nists and antagonists) have been shown to a¡ect mela-
tonin concentrations.32--34 Melatonin has been shown
to be metabolized by CYP1A2 in both animals and
humans.64,65 Thus, drugs known to a¡ect the activity
of CYP1A2 (ca¡eine, nicotine) and drugs metabolized
by CYP1A2 (e.g. £uvoxamine, 5-methoxypsoralen) are
likely to interfere with the metabolism of melatonin.
Early ¢ndings reporting high concentrations of melato-
nin following £uvoxamine66 and 5-methoxypsoralen67
administration can now be explained on the basis of
a CYP1A2-mediated mechanism. There is also some
preliminary evidence that the in£uence of oral contra-
ceptives on melatonin concentrations68,69 may occur at
the level of melatonin metabolism (unpublished data).
Further studies investigating this mechanism are
required.
Ideal sampling conditions
The correct procedure to collect blood, saliva and urine
for measurement of melatonin and 6-sulphatoxymela-
tonin is detailed in Table 1. Further details can also be
found on the Stockgrand Ltd website (www.stock-
grand.co.uk) and in a recent review.34
Circadian rhythm disorders are associated with sleep
problems. As such these conditions have been classi¢ed
in the International Classi¢cation of Sleep Disorders
(ICSD)70 as Circadian Rhythm Sleep Disorders. Circa-
dian rhythm sleep disorders may be the result of an
abrupt shift in time (e.g. as experienced following
transmeridian travel), or by an abrupt shift in sleep
and work time (as in rotating shift workers). Sleep dis-
turbance is caused by a mismatch between the internal
circadian timing system and the environment. Follow-
ing a period of re-adaptation, which depends on the
number of time zones crossed, synchrony is achieved
after time zone travel, but rarely in ‘normal’ night shift
work. Thus, most shift workers are living in a state of
intermittent, but chronic desynchrony.71,72 In unusual
circumstances (social isolation on o¡shore oil rigs
working certain schedules and during the polar win-
ter), most people can completely adapt their internal
clocks to night work.56,73 Circadian rhythm sleep disor-
ders may also be a result of circadian misalignment
such as advanced (ASPS) and delayed sleep phase
insomnia (DSPS), in which su¡erers have abnormally
early or late bedtimes, respectively. Apart from environ-
mental causes such as lighting at inappropriate times,
causes of ASPS and DSPS may be intrinsic and relate
to abnormal light sensitivity or aberrant clock timing
or both. Recent evidence suggests that polymorphisms
in a number of human clock genes (which could

Table 1 Sampling details for measurement of melatonin and 6-sulphatoxymelatonin (aMT6s)

Biological fluid Analyte Sampling procedure Precautions
Blood Melatonin Collect blood into heparinized tubes. Haemolysed plasma and plasma left in plastic
Centrifuge within 15 min. Store plasma at pipettes for more than 2–3 min may give

201C. falsely elevated melatonin levels.

Saliva Melatonin The best method is to ask subjects to spit
into polypropylene tubes. Do not stimulate
saliva production. Store at
201C.
Salivettes with an untreated cotton plug can
be used. Centrifuge for 15 min at 3000 r.p.m.
Store at
201C.
Do not eat within 30 min of sampling. Rinse
mouth with tap water before spitting. Saliva
left in plastic pipettes for more than 2–3 min
may give falsely elevated melatonin levels.
Use of salivettes may give falsely elevated
melatonin levels, standards should be run
through salivettes to correct for this.

Urine aMT6s Ask subjects to collect all urine passed over a
pre-set period into a standard urine bottle.
Measure and record the volume, store circa 5 mL
at
201C. Urine should be collected at least
every 3–4 h (longer during sleep period) for
at least 24 h, preferably for 48 h or longer.
For further details, see www.stockgrand.co.uk
Do not wash urine bottles with bleach or
another oxidant. No preservative is required
as aMT6s is stable in urine for 1 day at room
temperature, 2 days at 41C and for at least
2 years at
201C.

Ann Clin Biochem 2006; 43: 344–353

350 Skene and Arendt
feasibly a¡ect t) may be associated with ASPS
(per 2)74,75 and DSPS (per 3).76 Another less-common
circadian rhythm disorder is desynchronization from
the 24 h day as experienced most often in totally blind
people.5,6 There are also some reports of this non-24 h
sleep/wake disorder being observed in some sighted
people77 and in some dim-light, long-winter environ-
ments,78 although it is rarely seen on British Antarctic
Survey bases.79 Non-24 h sleep/wake disorder is cyclic,
characterized by periods of good sleep (long duration
sleep with no daytime napping) and periods of poor
sleep (short duration sleep with daytime naps36). The
periods of poor sleep have been shown to be associated
with abnormal timing of the circadian clock.23,36
Consequences
The physiological and behavioural consequences of cir-
cadian misalignment are known to anyone who has
travelled across time zones or who has done night shift
work: poor sleep of short duration with fatigue and
reduced alertness, vigilance and performance during
waking hours. These symptoms may predispose the
individual to accidents, error and risk. The long-term
consequences of repeated/continuous circadian desyn-
chrony such as may be experienced by shift workers are
just beginning to be studied. There is epidemiological
evidence to show increased cardiovascular risk80,81
and cancer risk82 in night shift workers. Eating meals
at night has been shown to produce increased blood tri-
glycerides and evidence of insulin resistance,83,84 both
risk factors for heart disease. Light exposure at night
has been hypothesized to increase cancer risk.85
Although it is always di⁄cult to relate cause and e¡ect
in chronic studies, these issues clearly require more
study.
Treatment strategies
The acute and possible long-term consequences of cir-
cadian rhythm disorders are driving the development
of treatment strategies to alleviate or at least minimize
some of the detrimental e¡ects of inappropriate circa-
dian timing (such as tiredness, reduced performance).
Currently, light and exogenous melatonins are the best
candidate treatments as both have been shown to
phase shift the timing of the human circadian clock.
Although other drug strategies have been used (e.g.
short acting benzodiazepines, moda¢nil, ca¡eine) to
alleviate some of the consequences of circadian misa-
lignment of jetlag and shift work, whether these drugs
act directly on the circadian clock is not yet clear. How-
ever, by modifying sleep and wakefulness, these drugs
indirectly a¡ect an individual’s light/dark exposure,
which, in turn, would shift the timing of the clock. It is
likely that the indirect e¡ect of these drugs on circadian
timing will form part of future treatment strategies.
Ann Clin Biochem 2006; 43: 344–353
However, side-e¡ects such as hangover and headache
will need careful monitoring.
Light
Light, appropriately timed, can advance or delay the
circadian timing system. Optimization of light’s phase-
shifting e¡ects is currently an intensive area of
research. It is clear that the extent to which light can
shift the clock is dependent upon the time of light
administration86,87 as well as the intensity of light,88
its duration and wavelength.53,89,90 Broadly speaking,
increasing the intensity and duration of the light sti-
mulus increases its e¡ect. Recent data have shown that
short wavelength light (420--480 nm) is most e¡ective
at phase shifting the human circadian clock.53,89,90 In
addition an individual’s light history (i.e. previous light
exposure) may be an important modulator of light’s
phase-shifting e¡ect. Direct proof of this is still out-
standing but it is predicted on the basis of reports show-
ing light history to a¡ect the ability of light to suppress
melatonin.91--93
Exogenous melatonin
Appropriately timed, exogenous melatonin has been
shown to advance or, more controversially, delay the
timing of the circadian clock.94,95 This phase-shifting
e¡ect of melatonin has been employed successfully to
entrain totally blind people with free-running circa-
dian rhythms.96,97 Entrainment by melatonin in our
studies has occurred by melatonin’s ability to phase
advance the circadian clock.96,98 In addition to correct-
ing the underlying desynchronized circadian disorder,
melatonin has also been shown to improve sleep and
reduce daytime napping in these subjects. However,
melatonin also improved sleep in those subjects that
did not entrain (but probably to a lesser extent). Melato-
nin’s phase-shifting e¡ect is presumably via receptors
in the SCN, although this is yet to be de¢nitely proven.
Current research is directed at determining the mini-
mum e¡ective dose for entrainment. Melatonin at a dai-
ly dose of 0.5 mg has been shown to e¡ectively entrain
free-running rhythms in blind people.98,99 At a single
dose of 0.05 mg, phase shifts can also be seen100 and
daily administration of this lower dose has recently
been tried in a single blind subject.101 Melatonin has
also been successful, when appropriately timed, in the
alleviation of DSPS, jet lag and in helping shift workers
to sleep during the day (reviewed by Arendt and
Skene26 and Arendt34).
Future perspectives
Although manipulation and optimization of the light-
ing environment remains key to minimizing the

detrimental e¡ect of circadian misalignment, the role
of non-photic stimuli in combination with lighting
requires further study. Timed exercise and meals
may reinforce the e¡ect of light and help maintain
entrainment. Melatonin is the treatment of choice for
non-24 h sleep/wake disorder experienced by the blind,
where light treatment is impossible. Further studies
determining the minimum e¡ective dose and the ideal
dosing regimen (daily/every second day) and formula-
tion (fast/slow release) are required. How melatonin’s
e¡ects are modulated by an individual’s t and pharma-
cokinetics is also not yet clear. Future lighting for
industry, institutions and the home needs to incorpo-
rate the recent research developments into practical
solutions. The e⁄cacy of lighting optimized on the basis
of the current research ¢ndings (e.g. optimal spectral
composition) now needs to be tested in the real-life
situation. In addition, the long-term risk of these
developments (e.g. blue light, daily melatonin) needs
careful monitoring.
Acknowledgements
The review was written during the tenure of an EU
Marie Curie RTN grant (MCRTN-CT-2004-512362) to
DJS, Health and Safety Executive/Energy Institute and
AFI (Antarctic Funding Initiative CGS-6-15) grants to JA.
Competing interests: DJS and JA are directors of Stock-
grand Ltd.
References
1 Middleton B, Arendt J, Stone BM. Human circadian rhythms in
constant dim light (8 lux) with knowledge of clock time. J Sleep
Res 1996; 5: 69–76
2 Hiddinga AE, Beersma DG, Van den Hoofdakker RH.
Endogenous and exogenous components in the circadian
variation of core body temperature in humans. J Sleep Res
1997; 6: 156–63
3 Wright Jr KP, Hughes RJ, Kronauer RE, Dijk DJ, Czeisler CA.
Intrinsic near-24-h pacemaker period determines limits of
circadian entrainment to a weak synchronizer in humans. Proc
Natl Acad Sci USA 2001; 98: 14027–32
4 Klerman EB, Rimmer DW, Dijk DJ, Kronauer RE, Rizzo III JF,
Czeisler CA. Nonphotic entrainment of the human circadian
pacemaker. Am J Physiol 1998; 274: R991–6
5 Lockley SW, Skene DJ, Arendt J, Tabandeh H, Bird AC, Defrance
R. Relationship between melatonin rhythms and visual loss in the
blind. J Clin Endocrinol Metab 1997; 82: 3763–70
6 Sack RL, Lewy AJ, Blood ML, Keith LD, Nakagawa H. Circadian
rhythm abnormalities in totally blind people: incidence and clinical
significance. J Clin Endocrinol Metab 1992; 75: 127–34
7 Skene DJ, Lockley SW, Hack LM, Arendt J. Free-running
circadian rhythms in the blind and their correction by melatonin
treatment. In: Honma K, Honma S, eds. Biological Rhythms.
Sapporo: Hokkaido University Press, 2005; 133–41
Human circadian rhythms 351
8 Duffy JF, Dijk DJ. Getting through to circadian oscillators: why use
constant routines? J Biol Rhythms 2002; 17: 4–13
9 Reppert SM, Weaver DR. Coordination of circadian timing in
mammals. Nature 2002; 418: 935–41
10 Hastings MH, Herzog ED. Clock genes, oscillators, and cellular
networks in the suprachiasmatic nuclei. J Biol Rhythms 2004; 19:
400–13
11 Glossop NR, Hardin PE. Central and peripheral circadian
oscillator mechanisms in flies and mammals. J Cell Sci 2002;
115: 3369–77
12 Yamazaki S, Straume M, Tei H, Sakaki Y, Menaker M, Block GD.
Effects of aging on central and peripheral mammalian clocks.
Proc Natl Acad Sci USA 2002; 99: 10801–6
13 Schibler U, Ripperger J, Brown SA. Peripheral circadian
oscillators in mammals: time and food. J Biol Rhythms 2003;
18: 250–60
14 Lucas RJ, Freedman MS, Munoz M, Garcia-Fernandez JM,
Foster RG. Regulation of the mammalian pineal by non-rod, non-
cone, ocular photoreceptors. Science 1999; 284: 505–7
15 Lucas RJ, Douglas RH, Foster RG. Characterization of an ocular
photopigment capable of driving pupillary constriction in mice.
Nat Neurosci 2001; 4: 621–6
16 Brainard GC, Hanifin JP, Greeson JM, et al. Action spectrum for
melatonin regulation in humans: evidence for a novel circadian
photoreceptor. J Neurosci 2001; 21: 6405–12
17 Thapan K, Arendt J, Skene DJ. An action spectrum for melatonin
suppression: evidence for a novel non-rod, non-cone photo-
receptor system in humans. J Physiol 2001; 535: 261–7
18 Provencio I, Jiang G, De Grip WJ, Hayes WP, Rollag MD.
Melanopsin: an opsin in melanophores, brain, and eye. Proc Natl
Acad Sci USA 1998; 95: 340–5
19 Berson DM, Dunn FA, Takao M. Phototransduction by retinal
ganglion cells that set the circadian clock. Science 2002; 295:
1070–3
20 Hattar S, Lucas RJ, Mrosovsky N, et al. Melanopsin and
rod–cone photoreceptive systems account for all major accessory
visual functions in mice. Nature 2003; 424: 76–81
21 Aschoff J, Fatranska M, Giedke H, Doerr P, Stamm D, Wisser H.
Human circadian rhythms in continuous darkness: entrainment
by social cues. Science 1971; 171: 213–5
22 Czeisler CA. The effect of light on the human circadian pace-
maker. Ciba Found Symp 1995; 183: 254–90; discussion 90–302.
23 Arendt J, Aldhous M, Wright J. Synchronisation of a disturbed
sleep–wake cycle in a blind man by melatonin treatment. Lancet
1988; 1: 772–3
24 Emens JS, Lewy AJ, Lefler BJ, Sack RL. Relative coordination
to unknown ‘weak zeitgebers’ in free-running blind individuals.
J Biol Rhythms 2005; 20: 159–67
25 Buxton OM, Lee CW, L’Hermite-Baleriaux M, Turek FW, Van
Cauter E. Exercise elicits phase shifts and acute alterations of
melatonin that vary with circadian phase. Am J Physiol Regul
Integr Comp Physiol 2003; 284: R714–24
26 Arendt J, Skene DJ. Melatonin as a chronobiotic. Sleep Med Rev
2005; 9: 25–39
27 Klein DC, Moore RY. Pineal N-acetyltransferase and hydro-
xyindole-O-methyltransferase: control by the retinohypothalamic
tract and the suprachiasmatic nucleus. Brain Res 1979; 174: 245–62
28 Perreau-Lenz S, Kalsbeek A, Garidou ML, et al. Suprachiasmatic
control of melatonin synthesis in rats: inhibitory and stimulatory
mechanisms. Eur J Neurosci 2003; 17: 221–8
Ann Clin Biochem 2006; 43: 344–353
352 Skene and Arendt
29 Perreau-Lenz S, Kalsbeek A, Pevet P, Buijs RM. Glutamatergic
clock output stimulates melatonin synthesis at night. Eur J
Neurosci 2004; 19: 318–24
30 Klein DC, Ganguly S, Coon SL, et al. 14-3-3 proteins in pineal
photoneuroendocrine transduction: how many roles? J Neuroen-
docrinol 2003; 15: 370–7
31 Simonneaux V, Ribelayga C. Generation of the melatonin
endocrine message in mammals: a review of the complex
regulation of melatonin synthesis by norepinephrine, peptides,
and other pineal transmitters. Pharmacol Rev 2003; 55: 325–95
32 Arendt J, Bojkowski C, Franey C, Wright J, Marks V.
Immunoassay of 6-hydroxymelatonin sulfate in human plasma
and urine: abolition of the urinary 24-h rhythm with atenolol.
J Clin Endocrinol Metab 1985; 60: 1166–73
33 Arendt J. Melatonin and the Mammalian Pineal Gland. London:
Chapman and Hall, 1995
34 Arendt J. Melatonin: characteristics, concerns, and prospects.
J Biol Rhythms 2005; 20: 291–303
35 Dijk DJ, Cajochen C. Melatonin and the circadian regulation of
sleep initiation, consolidation, structure, and the sleep EEG.
J Biol Rhythms 1997; 12: 627–35
36 Lockley SW, Skene DJ, Butler LJ, Arendt J. Sleep and activity
rhythms are related to circadian phase in the blind. Sleep 1999;
22: 616–23
37 Ebisawa T, Karne S, Lerner MR, Reppert SM. Expression
cloning of a high-affinity melatonin receptor from Xenopus
dermal melanophores. Proc Natl Acad Sci USA 1994; 91: 6133–7
38 Reppert SM, Godson C, Mahle CD, Weaver DR, Slaugenhaupt
SA, Gusella JF. Molecular characterization of a second mela-
tonin receptor expressed in human retina and brain: the Mel1b
melatonin receptor. Proc Natl Acad Sci USA 1995; 92: 8734–8
39 Reppert SM. Melatonin receptors: molecular biology of a new
family of G protein-coupled receptors. J Biol Rhythms 1997; 12:
528–31
40 Weaver DR, Stehle JH, Stopa EG, Reppert SM. Melatonin
receptors in human hypothalamus and pituitary: implications for
circadian and reproductive responses to melatonin. J Clin
Endocrinol Metab 1993; 76: 295–301
41 Weaver DR, Reppert SM. The Mel1a melatonin receptor gene is
expressed in human suprachiasmatic nuclei. NeuroReport 1996;
8: 109–12
42 Liu C, Weaver DR, Jin X, et al. Molecular dissection of two
distinct actions of melatonin on the suprachiasmatic circadian
clock. Neuron 1997; 19: 91–102
43 Dubocovich ML, Hudson RL, Sumaya IC, Masana MI, Manna E.
Effect of MT1 melatonin receptor deletion on melatonin-mediated
phase shift of circadian rhythms in the C57BL/6 mouse. J Pineal
Res 2005; 39: 113–20
44 Lincoln GA, Andersson H, Hazlerigg D. Clock genes and the long-
term regulation of prolactin secretion: evidence for a photoperiod/
circannual timer in the pars tuberalis. J Neuroendocrinol 2003; 15:
390–7
45 Maywood ES, Bittman EL, Hastings MH. Lesions of the
melatonin- and androgen-responsive tissue of the dorsomedial
nucleus of the hypothalamus block the gonadal response of male
Syrian hamsters to programmed infusions of melatonin. Biol
Reprod 1996; 54: 470–7
46 Waldhauser F, Lieberman HR, Lynch HJ, et al. A pharmacolo-
gical dose of melatonin increases PRL levels in males without
altering those of GH, LH, FSH, TSH, testosterone or cortisol.
Neuroendocrinology 1987; 46: 125–30
Ann Clin Biochem 2006; 43: 344–353
47 Ninomiya T, Iwatani N, Tomoda A, Miike T. Effects of exogenous
melatonin on pituitary hormones in humans. Clin Physiol 2001;
21: 292–9
48 Perras B, Ozcan S, Fehm HL, Born J. Melatonin does not inhibit
hypothalamic–pituitary–adrenal activity in waking young men.
J Neuroendocrinol 2005; 17: 811–6
49 Luboshitzky R, Levi M, Shen-Orr Z, Blumenfeld Z, Herer P, Lavie
P. Long-term melatonin administration does not alter pituitary–
gonadal hormone secretion in normal men. Hum Reprod 2000;
15: 60–5
50 Rajaratnam SM, Dijk DJ, Middleton B, Stone BM, Arendt J.
Melatonin phase-shifts human circadian rhythms with no
evidence of changes in the duration of endogenous melatonin
secretion or the 24-h production of reproductive hormones. J Clin
Endocrinol Metab 2003; 88: 4303–9
51 Arendt J. The Pineal Gland and Pineal Tumours. In: Grossman
A, section ed. Endotext.org, Online Endocrinology Textbook
2005; http://www.endotext.org/neuroendo/neuroendo15/neuro
endoframe15.htm
52 Arendt J. The Pineal Gland: Basic Physiology and Clinical
Implications. In: DeGroot LJ, Jameson JL, eds. Endocrinology.
Philadelphia: WB Saunders & Co, 2005
53 Warman VL, Dijk DJ, Warman GR, Arendt J, Skene DJ. Phase
advancing human circadian rhythms with short wavelength light.
Neurosci Lett 2003; 342: 37–40
54 Chemineau P, Daveau A, Bodin L, Zarazaga L, Gomez-Brunet
A, Malpaux B. Sheep as a mammalian model of genetic
variability in melatonin. Reprod Suppl 2002; 59: 181–90
55 Skene DJ, Lockley SW, Thapan K, Arendt J. Effects of light on
human circadian rhythms. Reprod Nutr Dev 1999; 39: 295–304
56 Barnes RG, Deacon SJ, Forbes MJ, Arendt J. Adaptation of the
6-sulphatoxymelatonin rhythm in shiftworkers on offshore oil
installations during a 2-week 12-h night shift. Neurosci Lett 1998;
241: 9–12
57 Gibbs M, Hampton S, Morgan L, Arendt J. Adaptation of the
circadian rhythm of 6-sulphatoxymelatonin to a shift schedule of
seven nights followed by seven days in offshore oil installation
workers. Neurosci Lett 2002; 325: 91–4
58 Skene DJ, Swaab DF. Melatonin rhythmicity: effect of age and
Alzheimer’s disease. Exp Gerontol 2003; 38: 199–206
59 Lewy AJ, Markey SP. Analysis of melatonin in human plasma by
gas chromatography negative chemical ionization mass spectro-
metry. Science 1978; 201: 741–3
60 Middleton B. Melatonin and 6-sulphatoxymelatonin. In: Wheeler
MJ, Morley Hutchinson JS, eds. Hormone Assays in Biological
Fluids. Totowa, NJ: Humana Press, 2005
61 Lewy AJ, Wehr TA, Goodwin FK, Newsome DA, Markey SP.
Light suppresses melatonin secretion in humans. Science 1980;
210: 1267–9
62 Bojkowski CJ, Aldhous ME, English J, et al. Suppression of
nocturnal plasma melatonin and 6-sulphatoxymelatonin by bright
and dim light in man. Horm Metab Res 1987; 19: 437–40
63 Deacon S, Arendt J. Posture influences melatonin concen-
trations in plasma and saliva in humans. Neurosci Lett 1994;
167: 191–4
64 Skene DJ, Papagiannidou E, Hashemi E, et al. Contribution of
CYP1A2 in the hepatic metabolism of melatonin: studies with
isolated microsomal preparations and liver slices. J Pineal Res
2001; 31: 333–42

65 Facciola G, Hidestrand M, von Bahr C, Tybring G. Cytochrome
P450 isoforms involved in melatonin metabolism in human liver
microsomes. Eur J Clin Pharmacol 2001; 56: 881–8
66 Skene DJ, Bojkowski CJ, Arendt J. Comparison of the effects of
acute fluvoxamine and desipramine administration on melatonin and
cortisol production in humans. Br J Clin Pharmacol 1994; 37: 181–6
67 Mauviard F, Raynaud F, Geoffriau M, Claustrat B, Pevet P. 5-
Methoxypsoralen inhibits 6-hydroxylation of melatonin in the rat.
Biol Signals 1995; 4: 32–41
68 Webley GE, Leidenberger F. The circadian pattern of melatonin
and its positive relationship with progesterone in women. J Clin
Endocrinol Metab 1986; 63: 323–8
69 Wright Jr KP, Myers BL, Plenzler SC, Drake CL, Badia P. Acute
effects of bright light and caffeine on nighttime melatonin and
temperature levels in women taking and not taking oral
contraceptives. Brain Res 2000; 873: 310–7
70 American Academy of Sleep Medicine. International Classification
of Sleep disorders: Diagnostic and Coding Manual. Westchester,
Illinois: American Academy of Sleep Medicine, 2005
71 Akerstedt T. Shift work and disturbed sleep/wakefulness. Sleep
Med Rev 1998; 2: 117–28
72 Rajaratnam SM, Arendt J. Health in a 24-h society. Lancet 2001;
358: 999–1005
73 Ross JK, Arendt J, Horne J, Haston W. Night-shift work in
Antarctica: sleep characteristics and bright light treatment.
Physiol Behav 1995; 57: 1169–74
74 Toh KL, Jones CR, He Y, et al. An hPer2 phosphorylation site
mutation in familial advanced sleep phase syndrome. Science
2001; 291: 1040–3
75 Carpen JD, Archer SN, Skene DJ, Smits M, von Schantz M. A
single-nucleotide polymorphism in the 50 -untranslated region of
the hPER2 gene is associated with diurnal preference. J Sleep
Res 2005; 14: 293–7
76 Archer SN, Robilliard DL, Skene DJ, et al. A length
polymorphism in the circadian clock gene Per3 is linked to
delayed sleep phase syndrome and extreme diurnal preference.
Sleep 2003; 26: 413–5
77 Hayakawa T, Uchiyama M, Kamei Y, et al. Clinical analyses of
sighted patients with non-24-h sleep-wake syndrome: a study of
57 consecutively diagnosed cases. Sleep 2005; 28: 945–52
78 Kennaway DJ, Van Dorp CF. Free-running rhythms of melatonin,
cortisol, electrolytes, and sleep in humans in Antarctica. Am J
Physiol 1991; 260: R1137–44
79 Broadway J, Arendt J, Folkard S. Bright light phase shifts the
human melatonin rhythm during the Antarctic winter. Neurosci
Lett 1987; 79: 185–9
80 Knutsson A, Akerstedt T, Jonsson BG, Orth-Gomer K. Increased risk
of ischaemic heart disease in shift workers. Lancet 1986; 2: 89–92
81 Kawachi I, Colditz GA, Stampfer MJ, et al. Prospective study of
shift work and risk of coronary heart disease in women.
Circulation 1995; 92: 3178–82
82 Schernhammer ES, Laden F, Speizer FE, et al. Rotating night
shifts and risk of breast cancer in women participating in the
nurses’ health study. J Natl Cancer Inst 2001; 93: 1563–8
83 Hampton SM, Morgan LM, Lawrence N, et al. Postprandial
hormone and metabolic responses in simulated shift work. J
Endocrinol 1996; 151: 259–67
Human circadian rhythms 353
84 Ribeiro DC, Hampton SM, Morgan L, Deacon S, Arendt J.
Altered postprandial hormone and metabolic responses in a
simulated shift work environment. J Endocrinol 1998; 158:
305–10
85 Davis S, Mirick DK, Stevens RG. Night shift work, light at night,
and risk of breast cancer. J Natl Cancer Inst 2001; 93: 1557–62
86 Minors DS, Waterhouse JM, Wirz-Justice A. A human phase-
response curve to light. Neurosci Lett 1991; 133: 36–40
87 Khalsa SB, Jewett ME, Cajochen C, Czeisler CA. A phase
response curve to single bright light pulses in human subjects.
J Physiol 2003; 549: 945–52
88 Boivin DB, Duffy JF, Kronauer RE, Czeisler CA. Dose–response
relationships for resetting of human circadian clock by light.
Nature 1996; 379: 540–2
89 Lockley SW, Brainard GC, Czeisler CA. High Sensitivity of the
human circadian melatonin rhythm to resetting by short
wavelength light. J Clin Endocrinol Metab 2003; 88: 4502–5
90 Revell VL, Arendt J, Terman M, Skene DJ. Short-wavelength
sensitivity of the human circadian system to phase-advancing
light. J Biol Rhythms 2005; 20: 270–2
91 Owen J, Arendt J. Melatonin suppression in human subjects by
bright and dim light in antarctica: time and season-dependent
effects. Neurosci Lett 1992; 137: 181–4
92 Hebert M, Martin SK, Lee C, Eastman CI. The effects of prior
light history on the suppression of melatonin by light in humans.
J Pineal Res 2002; 33: 198–203
93 Smith KA, Schoen MW, Czeisler CA. Adaptation of human pineal
melatonin suppression by recent photic history. J Clin Endocrinol
Metab 2004; 89: 3610–4
94 Lewy AJ, Bauer VK, Ahmed S, et al. The human phase response
curve (PRC) to melatonin is about 12 h out of phase with the
PRC to light. Chronobiol Int 1998; 15: 71–83
95 Wirz-Justice A, Werth E, Renz C, Muller S, Krauchi K. No
evidence for a phase delay in human circadian rhythms after a
single morning melatonin administration. J Pineal Res 2002; 32:
1–5
96 Lockley SW, Skene DJ, James K, Thapan K, Wright J, Arendt J.
Melatonin administration can entrain the free-running circadian
system of blind subjects. J Endocrinol 2000; 164: R1–6
97 Sack RL, Brandes RW, Kendall AR, Lewy AJ. Entrainment of
free-running circadian rhythms by melatonin in blind people. N
Engl J Med 2000; 343: 1070–7
98 Hack LM, Lockley SW, Arendt J, Skene DJ. The effects of low-
dose 0.5-mg melatonin on the free-running circadian rhythms of
blind subjects. J Biol Rhythms 2003; 18: 420–9
99 Lewy AJ, Bauer VK, Hasler BP, Kendall AR, Pires ML, Sack RL.
Capturing the circadian rhythms of free-running blind people with
0.5 mg melatonin. Brain Res 2001; 918: 96–100
100 Deacon S, Arendt J. Melatonin-induced temperature suppression
and its acute phase-shifting effects correlate in a dose-
dependent manner in humans. Brain Res 1995; 688: 77–85
101 Lewy AJ, Emens JS, Bernert RA, Lefler BJ. Eventual
entrainment of the human circadian pacemaker by melatonin is
independent of the circadian phase of treatment initiation: clinical
implications. J Biol Rhythms 2004; 19: 68–75
Accepted for publication 22 June 2006
Ann Clin Biochem 2006; 43: 344–353